Analysis of transcription in lens using tiled microarrays (ChIP on chip)

描述(申请人提供)：这项R21申请的长期目标是使用染色质免疫沉淀(ChLP)对晶状体中的基因表达进行综合探索性研究，然后使用NimbleGen(Chlps On Chip)开发的高密度平铺寡核苷酸阵列(ChIP)进行分析。这种方法将用于在2种模式生物--老鼠和鸡--的脊椎动物晶状体发育过程中，大幅加快数据收集、跨物种比较和调节网络的解剖。这一建议将(1)确定调控晶状体谱系形成的5个关键转录因子(Pax6和Sox2)以及分化(c-Maf、Prox1和Prb)与近100个编码晶状体发育和动态平衡所必需蛋白质的基因/位点的结合位点；以及(2)确定这些基因/位点的染色质结构，重点是组蛋白H3 K9乙酰化、H3 K3/27甲基化、DMA甲基化、绝缘蛋白CTCF和染色质激活剂HMGN3的结合，所有这些基因/位点都参与表观遗传调控。这些相互作用将在小鼠和鸡的晶状体中确定，每个基因座在10到500 kb之间，在定制的微阵列上平铺。这些基因座将包括所有晶状体结构基因(晶状体蛋白、膜和中间丝蛋白)、调控晶状体谱系形成的关键基因(如Pax6、Six3和Sox2)、其末端分化(Prox1、c-Maf和Sox1)以及在晶状体发育过程中活跃的信号转导网络的组成部分(如FGFR1至4)。这些数据将与RNA表达研究相关联，选定的数据将使用分子生物学实验进行验证。这些数据将为体内单个基因座远端调控区的功能鉴定、各种转录因子之间的交叉调控以及系统分析功能丧失和获得模型中晶状体的发育奠定基础。这些数据的产生和分析将用于新的聚焦R01项目，以阐明先天性晶状体缺陷和白内障期间的正常晶状体发育和异常基因调控。

DESCRIPTION (provided by applicant): The long-term goal of this R21 application is to perform integrative exploratory studies of gene expression in lens using chromatin immunoprecipitations (ChlPs) followed by analysis using high-density tiled oligonucleotide arrays (chips) developed by Nimblegen (ChlPs on chip). This approach will be used for massive acceleration of data collection, cross-species comparisons and dissection of regulatory networks during vertebrate lens development in 2 model organisms, mouse and chicken. This proposal will (1) Determine the profiles of binding sites of 5 key transcription factors regulating lens lineage formation, Pax6 and Sox2, and differentiation (c-Maf, Proxl and pRb) with nearly 100 genes/loci encoding proteins essential for lens development and homeostasis; and (2) Identify chromatin structure of these genes/loci focusing on histone H3 K9 acetylation, H3 K3/27 methylation, methylation of DMA, binding of insulator protein CTCF and chromatin activator HMGN3, all involved in epigenetic regulation. These interactions will be determined in mouse and chicken lens with each locus between 10 to 500 kb, tiled on custom-based microarrays. These loci will include all lens structural genes (crystallins, membrane and intermediate filament proteins), key genes regulating lens lineage formation (e.g., Pax6, Six3 and Sox2), its terminal differentiation (Proxl, c-Maf and Sox1) and components of signal transduction networks (e.g., FGFR1 to 4) active during lens development. The data will be correlated with RNA expression studies and selected data will be validated using molecular biology experiments. This data will lay a foundation for the functional identification of distal regulatory regions of individual loci in vivo, identification of cross-regulation between various transcription factors, and systemic analysis of lens development in loss-of-and gain-of-function models. Generation and analysis of this data will be used for new focused R01 projects to elucidate normal lens development and abnormal gene regulation during congenital lens defects and in cataracts.